PROJECT SUMMARY Despite major advances in the treatments, diarrheal diseases are a major health burden worldwide. Although oral rehydration therapy has significantly reduced mortality from infectious diarrhea, more efficacious drugs are needed to reduce morbidity and mortality caused by enteric infections. Activation of aryl hydrocarbon receptor (AhR) has been shown to be an effective therapeutic strategy to combat enteric infections and other forms of gastrointestinal inflammation. AhR is a ligand-activated nuclear receptor that coordinates anti-microbial responses in the gut, in addition to xenobiotic detoxification. Activation of AhR by exogenous compounds has been well established, while its activation by endogenous molecules warrants further investigation. Our preliminary data showed that serotonin (5-hydroxytryptamine, 5-HT), a tryptophan derived important neurotransmitter and hormone, could serve as an endogenous ligand/activator of AhR in intestinal epithelial cells. 5-HT is internalized in the cell via its uptake through a Na+/Cl- dependent serotonin transporter (SERT). Interestingly, our studies demonstrated that 5-HT increases the expression of CYP1A1, the canonical gene target of AhR, via a SERT dependent process. Additionally, the expression of CYP1A1 and of CCL20 was suppressed in the intestinal mucosa of mice lacking functional serotonin transporter (SERT). CCL20, an additional AhR gene target, is an anti-microbial peptide produced by epithelial cells that attracts immune cells to sites of infection. Thus, to establish the novel link between 5-HT and AhR dependent pathways, our proposed studies will test the hypothesis that 5-HT or its metabolites act as endogenous modulators of AhR. We will further examine if decreased uptake of 5-HT by SERT deficiency leads to impaired bacterial defenses. Studies proposed in Specific Aim 1 will investigate the molecular mechanisms of AhR activation by 5-HT using state-of-art in vitro and ex vivo models including intestinal epithelial cell lines and enteroids that faithfully recapitulate the crypt-villus architecture of the intestine. Specific Aim 2 will examine the susceptibility of SERT KO mice to Citrobacter rodentium infection, a mouse pathogen closely related to clinically important human pathogens Enteropathogenic E.coli and Enterohemorrhagic E. coli as a model of infectious disease. To confirm the role of SERT in AhR modulated pathways, we will evaluate the ability of transgenic mice with intestinal specific overexpression of SERT to upregulate AhR gene targets involved in the antimicrobial responses in the absence or presence of bacterial lipopolysaccharise (LPS). Overall, the outcome of the proposed studies will demonstrate the importance of intestinal 5-HT and SERT as novel therapeutic targets to treat gut disorders such as enteric infections or inflammation. These studies will also form a critical training platform for the PI's growth as an inquisitive scientist and as a skilled clinician.